Molecular snapshots of the Pex1/6 AAA + complex in action

The peroxisomal proteins Pex1 and Pex6 form a heterohexameric type II AAA+ ATPase complex, which fuels essential protein transport across peroxisomal membranes. Mutations in either ATPase in humans can lead to severe peroxisomal disorders and early death. We present an extensive structural and biochemical analysis of the yeast Pex1/6 complex. The heterohexamer forms a trimer of Pex1/6 dimers with a triangular geometry that is atypical for AAA+ complexes. While the C-terminal nucleotide-binding domains (D2) of Pex6 constitute the main ATPase activity of the complex, both D2 harbour essential sub-strate-binding motifs. ATP hydrolysis results in a pumping motion of the complex, suggesting that Pex1/6 function involves substrate translocation through its central channel. Mutation of the Walker B motif in one D2 domain leads to ATP hydrolysis in the neighbouring domain, giving structural insights into inter-domain communication of these unique heterohexameric AAA + assemblies

The Peroxisomal Targeting Signal 3 (PTS3) of the Budding Yeast Acyl-CoA Oxidase Is a Signal Patch

The specificity of import of peroxisomal matrix proteins is dependent on the targeting signals encoded within their amino acid sequences. Two known import signals, peroxisomal targeting signal 1 (PTS1), positioned at the C-termini and PTS2 located close to N-termini of these proteins are recognized by the Pex5p and Pex7p receptors, respectively. However, in several yeast species, including Saccharomyces cerevisiae, proteins exist that are efficiently imported into peroxisomes despite having neither PTS1 nor PTS2 and for which no other import signal has been determined. An example of such a protein is S. cerevisiae acyl-CoA oxidase (AOx) encoded by the POX1 gene. While it is known that its import is driven by its interaction with the N-terminal segment of Pex5p, which is separate from its C-terminal PTS1-recognizing tetratricopeptide domain, to date, no AOx polypeptide region has been implicated as critical for this interaction, and thus would constitute the long-sought PTS3 signal. Using random mutagenesis combined with a two-hybrid screen, we identified single amino acid residues within the AOx polypeptide that are crucial for this interaction and for the peroxisomal import of this protein. Interestingly, while scattered throughout the primary sequence, these amino acids come close to each other within two domains of the folded AOx. Although the role of one or both of these regions as the PTS3 signal is not finally proven, our data indicate that the signal guiding AOx into peroxisomal matrix is not a linear sequence but a signal patch